Elastomeric polypeptide matrices for preventing adhesion of biological materials

ABSTRACT

The invention provides a bioelastomer comprising tetrapeptide and/or pentapeptide monomeric units of the formula: 
     
         R.sub.1 PGR.sub.2 G.sub.n 
    
     wherein R 2  is a peptide-producing residue of alanine or glycine; P is a peptide-producing residue of proline; G is a peptide-producing residue of glycine; R 2  is a peptide-producing residue of glycine or alanine; and n is 0 or 1. In a further aspect of the invention, a method is provided for preventing adhesion of biological materials, such as protein, cells, and tissues, by forming a protective layer between a first surface and a second surface using the bioelastomer.

This invention as made with U.S. Government support under contractN00014-90-C-0265 awarded by the Department of the Navy. The Governmenthas certain rights in this invention.

INTRODUCTION

1. Technical Field

The present invention relates to the field of biocompatible structuralmaterials and coatings which are suitable for in vivo applications.

2. Background

Bioelastomeric polypeptides are a relatively new development that arosein the laboratories of the present inventor and are disclosed in aseries of previously fried patents and patent applications. For example,U.S. Pat. No. 4,474,851 describes a number of tetrapeptide andpentapeptide repeating units that can be used to form a bioelasticpolymer. Specific bioelastic polymers are also described in U.S. Pat.Nos. 4,132,746, 4,187,852, 4,589,882, and 4,870,055. U.S. Pat. No.5,064,430 describes polynonapeptide bioelastomers. Bioelastic polymersare also disclosed in related patents directed to polymers containingpeptide repeating units that are prepared for other purposes but whichcan also contain bioelastic segments in the final polymer: U.S. Pat.Nos. 4,605,413, 4,976,734, and 4,693,718, entitled "Stimulation ofChemotaxis by Chemotactic Peptides"; U.S. Pat. No. 4,898,926, entitled"Bioelastomer Containing Tetra/Pentapeptide Units"; U.S. Pat. No.4,783,523 entitled "Temperature Correlated Force and StructureDevelopment of Elastin Polytetrapeptide"; U.S. Pat. Nos. 5,032,271,5,085,055, and 5,255,518, entitled "Reversible Mechanochemical EnginesComprised of Bioelastomers Capable of Modulable Temperature Transitionsfor the Interconversion of Chemical and Mechanical Work"; U.S. Pat. No.4,500,700, entitled "Elastomeric Composite Material Comprising aPolypeptide"; and U.S. Pat. No. 5,250,516 entitled "BioelastomericMaterials Suitable for the Protection of Wound Repair Sites." A numberof other bioelastic materials and methods for their use are described inpending U.S. patent applications including: U.S. Ser. No. 07/184,873,filed Apr. 22, 1988, now U.S. Pat. No. 5,336,256 entitled "ElastomericPolypeptides as Vascular Prosthetic Materials"; U.S. Serial No.07/962,608, filed Oct. 16, 1992, now abandoned entitled "BioelastomericDrug Delivery System"; and U.S. Ser. No. 08/187,441, filed Jan. 24,1994, entitled "Photoresponsive Polymers". All of these patents andpatent applications are herein incorporated by reference, as theydescribe in detail bioelastomers and/or components thereof and theirpreparation. This information can be used in preparing and using thecompositions and methods of the present invention, which are preparedfrom different specific monomeric units but can be prepared (and in manycases used, although with the advantages described herein) in the samemanner as previously prepared bioelastomers.

The prior art bioelastic materials have been proposed for a number ofuses and apparatuses, as indicated by the general subject matter of theapplications and patents set forth above. The bioelastic compositionsand machines respond to pressure, chemical, light, and/or thermalchanges in the environment by phase transitions (e.g., viscosity orturbidity changes) or by contraction or relaxation to reversiblytransduce these energies into mechanical work (for example, as describedin U.S. Pat. No. 5,226,292).

The bioelastomers were developed based on investigations into thenatural bioelastomer elastin. Elastin is comprised of a single proteincontaining a serial alignment of alaninc-rich, lysine-containingcross-linking sequences alternating with glycine-rich hydrophobicsequences. With the entire bovine sequence known, the most strikinghydrophobic sequences, both from the standpoint of length and ofcomposition, are one that contains a polypentapeptide (PPP) and one thatcontains a polyhexapeptide (PHP). Elastin also contains severaltetrapeptide (TP) units. As a result of work conducted by the presentinventors, the polypentapeptide of elastin when cross-linked has beenfound to be elastomeric and the polyhexapeptide thereof has been foundto be non-elastomeric and appears to provide a means for aligning andinterlocking the chains during elastogenesis. It has also been foundthat the elastin polypentapeptide and polytetrapeptide are bothconformation-based elastomers that develop entropic elasticity andstrength on undergoing an inverse temperature transition to form aregular β-turn containing dynamic structure.

A typical biological elastic fiber is comprised of a large elastin corecovered with a fine surface layer of microfibrillar protein. Elastin isformed upon cross-linking of the lysine residues of tropoelastin. Therepeating elastin pentapeptide has the formula (VPGVG)_(n), while therepeating hexapeptide has the formula (VAPGVG)_(n), where n variesdepending upon the species. The tetrapeptide unit has the formula(VPGG). These sequences, of course, utilize the standard one-letterabbreviation for the constituent amino acids.

It has been found that these polypeptides are soluble in water below 25°C. but on raising the temperature they associate reversibly to form awater-containing viscoelastic phase in the polypentapeptide (PPP) andpolytetrapeptide (PTP) cases, whereas in the polyhexapeptide (PHP) case,they associate irreversibly in water to form a granular precipitate,which usually requires the addition of trifluoroethanol to the aggregatefor redissolution. On cross-linking, the former (PPP) and (PTP) havebeen found to be elastomers, whereas PHP is not elastomeric.

For purposes of clarification, it is noted that the reversibletemperature elicited aggregation, which gives rise upon standing to adense viscoelastic phase, is called coacervation. The viscoelastic phaseis called the coacervate, and the solution above the coacervate isreferred to as the equilibrium solution.

Most importantly, cross-linked PPP, PTP and analogs thereof at fixedlength exhibit elastomeric force development at different temperaturesspanning a range of up to about 75° C. depending upon severalcontrollable variables. Moreover, these cross-linked elastomers developnear maximum elastomeric force over a relatively narrow temperaturerange. Thus, by synthesizing bioelastomeric materials having varyingmolar amounts of the constituent pentamers and tetramers together withsuch units modified by hexameric repeating units, and by choosing aparticular solvent to support the initial viscoelastic phase, it ispossible to rigorously control the temperature at which the obtainedbioelastomer develops elastomeric force.

In general, the process of raising the temperature to form the aboveelastomeric state is an inverse temperature transition resulting in thedevelopment of a regular non-random structure, unlike typical rubbers,which utilizes, as a characteristic component, hydrophobicintramolecular interactions. The regular structure is proposed to be aβ-spiral, a loose water-containing helical structure with β-turns andspacers between turns of the helix which provides hydrophobic contactsbetween helical turns and has suspended peptide segments. These peptidesegments are free to undergo large amplitude, low frequency rockingmotions called librations. This mechanism of elasticity is called thelibrational entropy mechanism of elasticity (or is sometimes referred toas resulting from damping of internal chain dynamics on extension). Theelastomeric force of these various bioelastomers develops as the regularstructure thereof develops. Further, a loss of regular structure by hightemperature denaturation results in loss of elastomeric force. Thesepolymers can be prepared with widely different water compositions, witha wide range of hydrophobicities, with almost any desired shape andporosity, and with a variable degree of cross-linking by selectingdifferent amino acids for the different positions of the monomeric unitsand by varying the cross-linking process, e.g. chemical, photochemical,enzymatic, irradiative, used to form the final product.

The bioelastomeric polymers have considerable potential for use inmedical and other applications, as they can be modified in structure toprovide a number of different biological properties. One type of desiredproperty, which has been previously achieved to some extent, is lowadhesion of proteins, cells, and other biological components. Adhesionsaccompanying the healing of wounds, whether due to surgery or othertrauma, give rise to many disadvantageous effects. For example,peritoneal cavity adhesions after surgery lead to intestinal obstructionand necessitate recurring operations. Furthermore unwanted adhesionsthemselves pose problems during recurrent operations. In anotherexample, tendon adhesions often compromise tendon surgery and repair.Still again, adhesion often arise after heart by-pass surgery, sometimeseven resulting in adhesion of heart muscle to the back of the breastbone. Pericardial adhesions often arise in heart surgery. In biologicalsituations involving implantation of exogenous material into a tissue,as in the use of permanent artificial joints or temporary implants suchas catheters, surgical drains, and shunts, cell adhesion and resultingcell growth can impair the functioning of the implant or of thesurrounding tissue. Clearly, it would be advantageous to find a suitablematerial that could function as a perfect insulator material forisolating wound repair sites from adhesions whether between layers ofabdominal and thoracic walls, between repair sites within the abdomen orthorax, within the hand, wrist, foot, ankle, and other joints, orbetween the skin and body stroma, as well as for protecting implantedmaterials from stimulating cell adhesion. Such a material would have tosatisfy many prerequisites. For example, it would be necessary that sucha material would match the compliance of the soft tissue site ofapplication. The material would also need to be biologically inert (orat least degradable to non-toxic products) and be obtainable indifferent forms, such as elastomeric sheets, foams or powders, thatwould provide sufficient ease of handling for each particularapplication. Of course, such a material would also have to be readilysterilizable as well as being biocompatible and eliciting insignificantimmunogenic and antigenic responses in the host. By biologicalcompatible is meant that the material in final form will not harm theorganism or cell into which it is implanted to such a degree thatimplantation is as harmful or more harmful than absence of treatmentwith the indicated material. The same properties described above wouldalso be advantageous in a material for the protection of bum areas andto facilitate repair of the damaged tissue.

Materials exhibiting these properties have been described in U.S. Pat.No. 5,250,516. However, room for improvement remains, and it would bebeneficial to provide a material for medical applications that fullymeets all of the above requirements and furthermore provides a surfaceto which biological materials, whether cells or macromolecules such asproteins or polynucleotides, do not adhere in an in vivo or in vitrosituation, especially one in which blood or a blood component such asserum is present.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide anelastomeric material which is compatible with and similar to the softtissue at wound sites and which is also biodegradable, if at all, tonon-toxic components.

It is also an object of the invention to provide a material that isavailable in different physical forms, such as sheets, gels, foams, orpowders, or that can be grafted onto another surface to provide ease ofhandling for various applications.

It is also an object of the invention to provide a process for isolatingwound repair sites or implants to effect a more salutary wound healingprocess.

According to the present invention, the foregoing and other objects areobtained by providing an improved biocompatible material comprising apolymer made from tetrapeptide and or pentapeptide monomers of theformula R₁ PGR₂ G_(n) wherein R₁ is a peptide-producing residue ofalanine or glycine; P is a peptide-producing residue of proline; G is apeptide-producing residue of glycine; R₂ is a peptide-producing residueof glycine or alanine; and n is 0 or 1. The polymer can be present as acopolymer containing a mixture of tetrameric and pentameric units andfurther can contain other monomeric units in some embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood by reference to the followingdetailed description of specific embodiments together with the figuresthat form part of this specification, wherein:

FIG. 1 is a schematic diagram showing the mean number of LNFs and HUVECsadhering to X²⁰ -poly(GGAP), X²⁰ -poly(GGVP), X²⁰ -poly(GGIP), and X²⁰-poly(GVGVP) matrices after 3 h in the presence of 0.1% BSA. TC-O andTC-hFN represent uncoated and human fibronectin coated (10 μg/mL) tissueculture plastic substrata. The vertical bars represent one standarderror.

FIG. 2 is a schematic diagram showing the mean number of LNFs and HUVECsadhering to X²⁰ -poly(GGAP), X²⁰ -poly(GGVP), X²⁰ -poly(GGIP), and X²⁰-(GVGVP) matrices after 3 h in the presence of 10% fetal bovine serum(LNFs) and 20% fetal bovine serum (HUVECs). TC represents normal tissueculture plastic substratum. The vertical bars represent one standarderror.

FIG. 3 is a schematic diagram showing the mean number of LNFs and HUVECsadhering to X²⁰ -poly(GGAP), X²⁰ -poly(GGVP), X²⁰ -poly(GGIP), and X²⁰-poly(GVGVP) matrices after 20 h in the presence of 10% fetal bovineserum (LNFs) and 20% fetal bovine serum (HUVECs). TC represents normaltissue culture plastic substratum. The vertical bars represent onestandard error.

DESCRIPTION OF SPECIFIC EMBODIMENTS

In its broadest aspect, the bioelastomers of the present inventioncomprise tetrapeptide and/or pentapeptide units of the formula R₁ PGR₂G_(n) where R₁ and R₂ are peptide-producing residues of alanine orglycine, P is a peptide-producing residue of proline, G is apeptide-producing residue of glycine, and n is 0 or 1. These units aregenerally present in the polymer in an amount sufficient to provideelastomeric properties and to adjust the development of elastomericforce of the bioelastomer to a predetermined temperature, as has beendescribed for previous bioelastomers.

These polymers differ from specific polymers previously disclosed in thelaboratories of the inventor in several important, although subtle,ways. For example, it was previously discovered and reported that apolymer prepared from the repeating pentameric unit --APGVG-- did nothave elastomeric properties but was instead granular, a property thatruled out the use of such polymers in the types of situationscontemplated for the polymers of the present invention. This was thoughtto result from the presence of an alanine residue at position 1 of theβ-turn, which was thought to disrupt this structure that is essentialfor elasticity. However, it now appears as a result of theinvestigations leading to the present invention that an alanine (orglycine) can be present at position 1 without disrupting the β-turn ifthere is an alanine or glycine present at position 4. Additionally, thepresence of an alanine or glycine at position 4, instead of a morehydrophobic amino acid residue, such as valine or isoleucine, is usefulin preventing the adhesion of macromolecular species such as proteinsand nucleic acids to surfaces prepared from bioelastic polymers.Adhesion of such biological macromolecules and other biologicalmaterials to a surface is often preliminary to adhesion of cells andtissues. Thus the polymers of the invention are useful in form barriersto cell and tissue adhesion in a number of biological situations.

There appears to be no upper limit to the molecular weight of usefulpolymers of the invention except that imposed by the processes of makingthese polymers. Polymers containing up to about 250 pentamers have beensynthesized in E. coli using recombinant DNA methods. Typical polymerscontain at least 5, preferably at least 10, more preferably at least 20,tetrapeptide or pentapeptide monomers, and because of poor solubility inaqueous solvents, which are desirable for biological uses, usuallycontain fewer than 1000, usually fewer than 500, of such units.

It should be noted that bioelastomeric polypeptide chains containing therepeating units can have tetrapeptide or pentapeptide "monomers" thatare permutations of the basic sequence (e.g., poly-GGAP vs. poly-APGG).If the polymer is not synthesized using the pentapeptide "monomers", butrather is synthesized by sequential adding of amino acids to a growingpeptide, such as in the case of an automatic peptide synthesizer or ingenetic production, the designation of the repeating unit is somewhatarbitrary. Moreover, "incomplete units" can flank regions of, forexample, a polytetramer so that GG-(APGG)₁₀ -A may equally well bethought of as G-(GAPG)₁₀ -GA or (GGAP)₁₀ -GGA). Designation of amaterial as, for example, poly-WXYZ is therefore intended to encompassall same-sequence permutations (poly-XYZW, poly-YZWX, and poly-ZWXY),unless otherwise stated or clear from the context.

The bioelastomer can be uncross-linked or cross-linked, depending on themanner of its ultimate use. For example, if the bioelastomer is used asa surface coating on a second material that provides appropriatemechanical properties, cross-linking is not necessary to provedmechanical strength. Cross-linking provides mechanical strength andrigidity to the polymer, and increasing amounts of cross-linking areappropriate for increasing demands of rigidity. Typical amounts ofcross-linking provide one cross-link for every 5-100 tetrapeptide orpentapeptide units. Methods for cross-linking bioelastomericpolypeptides are known in the art. For example, U.S. Pat. No. 4,589,882,incorporated herein by reference, teaches enzymatic cross-linking bysynthesizing block polymers having enzymatically cross-linkable units.These bioelastic polymers are described in the various patents and otherdocuments listed above that arose in the laboratories of the presentinventors. Additionally, cross-linking by irradiation is described indetail in nearly all of the prior patents arising from the laboratoriesof the inventor, which have been incorporated by reference above.Further specific examples are set out below. Polymers described in thisspecification that are prepared by irradiation cross-linking areidentified as, for example, "X²⁰ -poly(GGAP)," which refers to a polymerprepared from GGAP tetrapeptide units that has been γ-irradiated with a20 Mrad dose of cobalt-60 radiation to form the cross-links which resultin an insoluble matrix.

In order to obtain high molecular weight polymers in good yields, anumber of approaches are available. When producing polymers by chemicalsynthesis, care should be taken to avoid impurities, because smalllevels of impurities can result in termination of the polymerizationprocess or in racemerization that can alter the physical properties ofthe resulting polymer, but there are otherwise no particular problems ofsynthesis. Different bioelastomer unit permutations have been preparedand polymerized with different coupling methods, and one such techniqueis described in detail in the examples below. Peptide unit purity isimportant in obtaining a material with suitable physical properties,since small changes in the preparation of the pentamers can result in atransition temperature that varies as much as 15° C. (25° C.-40° C.).This variance is important to consider since a polymer that has a 25° C.transition temperature will form a very good cross-linked elastomericmatrix, while a preparation having a 40° C. transition temperature willnot cross-link to form an elastomeric matrix. The solution of thispotential problem is simply to purify the components used to prepare thepeptide.

Synthesis of the bioelastomeric repeating units of the present inventionis straightforward and easily accomplished by a peptide chemist or bystandard methods in microbial fermentation. In order to preparepoly(GGXP), where X is Gly, Ala, Val or Ile, several different syntheticcoupling strategies have been used. In one approach, a 2+2 strategy wasused in which the dipeptides Gly-Gly and X-Pro were coupled together,while in a second approach, a step-wise addition strategy starting atthe C terminus was used. The step-wise addition approach gave a betteryield and a product with a lower transition temperature as compared tothe 2+2 approach.

An alternative to the organic synthesis of protein-based polymers is abiosynthetic approach using current recombinant DNA methodologies. Usingthis approach, a gene encoding the desired peptide sequence isconstructed, artificially inserted into, and then translated in amicrobial host organism. The resulting protein can then be purified,often in large amounts, from cultures grown in fermentation reactors.Molecular biology techniques known in the art are used to manipulate thegenetic information (i.e., DNA sequences) for their effective expressionin the appropriate host organism (see, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989)). The primary tools that make this possible areknown in the art and include enzymes capable of cleaving, joining,copying and otherwise modifying polynucleotides. In addition, vectorsallowing the introduction of this information into the host organism ina suitable manner for expression are also known in the art. A detailedexample of the production of poly-VPGVG is set out in McPherson et al.,"Production and Purification of a Recombinant Elastomeric Polypeptide,G-(VPGVG)₁₉ -VPGV, from Escherichia coli," Biotechnol. Prog.,1992:347-352, a publication arising from the laboratory of the presentinventor. This publication can be used as guidance for genetic-basedproduction of the material of the present invention.

The basic polymer of the invention, in its simplest form, is prepared asa homopolymer from one of the eight basic monomers, namely APGA, APGG,GPGA, GPGG, APGAG, APGGG, GPGAG, and APGGG. Of these, polymers formedfrom tetrapeptide units or from units in which the first amino acidresidue is alanine are preferred, particularly those formed from APGG.Either random or block copolymers prepared from the monomeric units arealso useful for the indicated purposes but are less preferred when anequivalent homopolymer has the desired physical properties, simplybecause of the greater complexity of synthesis.

In some embodiments the present invention uses the specifiedtetrapeptide and pentapeptide units to form a matrix which is thenmodified in a variety of ways to obtain additional properties. A matrixformed from the indicated monomeric units is particularly useful forbiological applications, since the biological inertness of the matrixprovides an excellent background in which other activities can be eithermaximally effected or measured. Considerable variations in the aminoacids that are present at various locations in the resulting polymer ispossible as long as the multiple β-turns with intervening suspendedbridging segments are retained in order to preserve elasticity and thesequences do not promote adhesion more than is desired for a particularapplication. For this reason it is preferred that at least 50% of thepolypeptide is formed from the indicated monomeric units, morepreferably at least 70%, even more preferably at least 90%.Nevertheless, it is possible to prepare polypeptides in which thesemothomeric units are interspersed throughout a larger polypeptide thatcontains peptide segments designed for other purposes. For example, thebioelastomer can contain naturally occurring sequences which arecomponents of connective tissue. These can be insertions of, forexample, single amino acids between monomeric units, substitutions ofone amino acid for another in an occasional monomer, or inclusion ofdifferent polypentapeptide, polyhexapeptide or polytetrapeptidesequences which can be added either in parallel or in sequence toincrease strength, elastic modulus and ease of handling. The bioelasticunits of the invention can be attached to or interspersed among othertypes of molecules, which molecular units can impart functions to thepolymer such as biological activity, chemotaxis, protease, or nucleasesusceptibility. Such molecules include peptides, proteins, nucleic acid,DNA, RNA, carbohydrates and lipid chains. As disclosed in earlier U.S.Patents, additional properties, e.g. strength, specific binding, areimported to bioelastomeric materials by compounding the repeatingelastic units to a second material with greater strength or with thedesired property as disclosed in U.S. Pat. Nos. 4,474,851 and 5,064,430.Such compounding can be oriented in the backbone of the polymer bypreparing copolymers in which bioelastic units that form β-turns areinterspersed among polymer units providing a desired property e.g. celladhesion sequences for appropriate tissue cells. These sequences can beadded covalently and sequentially or as side chains to provide for thedesired cell adhesion (or lack thereof) as in a tendon sheath or in afascia or in bum covers. The ratio of these other sequences to themonomer residue can range from 1:2 to 1:5000. Preferably the ratio is1:10 to 1:100. The upper limit on the number and kind of substituents isalso influenced by the ability of the elastic polymer to fold/assembleproperly to attain a beta-spiral in the relaxed state.

When considering bioelastomers of the invention, it will be apparentthat artificial materials are intended and that there is no intention toclaim elastin or other natural materials. As discussed previously forbioelastomers, the defined structure of the artificial bioelastomers ofthe invention allow full and complete design of the physical propertiesof the bioelastomers, rather than having to rely on the lesscontrollable properties that would exist for material prepared fromnatural products.

The location of a random or systematic substituent in the polymer, withrespect to the monomer residue side-chain position, is not critical solong as the beta-turn is not prevented from forming in the relaxedstate. Preferred positions for the various peptides of the invention areas taught in the patents and pending applications from the laboratory ofthe present inventor in this area, which have been incorporated byreference.

In addition, additional amino acid residues can be optionallyinterspersed within the polymer to enable covalent linkage of thebioelastic polymer to a surface, although simple coating of a surfacewith a solution of the polymer is satisfactory for many situations,especially when only short-term protection of the surface is necessary.For example, cysteine can be introduced into the polymer to allow forlinkage via disulfide bridges to a surface or lysine can be introducedfor enzymatic linkage to a surface. In a preferred embodiment, one ormore of these linking groups are present at one or both of the terminalends of a polypeptide strand, either at the ultimate terminus or withinthe 5 % of residues at the terminus, rather than in the interior of apolypeptide strand. When the polymer is prepared using genetictechniques, the reactive linking groups are preferably at the N-terminalportion of the molecule. In such embodiments, covalent attachment occursbetween a functional group in the bioelastomer and a functional group inthe material that forms the surface, which can itself be a differentbioelastomer, such as those described in the prior art. Attachment ofbioelastomers to surfaces and various coating processes for surfaces aredescribed in various of the patents cited in the Background section ofthis specification and are not essential parts of the present invention.

As discussed supra, it has been found that the elastic polypentapeptideand polytetrapeptide bioelastomers are both conformation-basedelastomers that develop entropic elasticity and strength on undergoingan inverse temperature transition to form a regular β-turn containingdynamic structure. In the presence of serum, considerable but submaximalcell attachment occurs using bioelastomers known in the art, e.g., X²⁰-poly(GVGVP), poly(GGIP), and poly(GGVP), many of which, particularlypoly(GGVP), are quite close in structure to the polymers of the presentinvention. In contrast, however, the present bioelastomers result in nocell attachment even in the presence of serum, making the bioelastomersof the invention prepared from the selected group of monomers a moreeffective physical barrier for prevention of adhesions than thepreviously used materials.

The elastomeric polypeptides of the present invention have the abilityto control adhesions at wound repair sites even in the presence ofserum. Moreover, the polypeptides are biodegradable and biocompatible asa soft tissue implant, are readily sterilizable, and furthermore can beformed in cross-linked sheets or strips, varying from a gelatinous to ateflon-like consistency. It is even possible to prepare these materialsin a deformable foam-like state with or without cross-linking. Also,when efficacious, the sequential polypeptide could be applied as apowder, which on absorbing water produces a sticky viscoelastic gel-likematerial.

In the presence of much serum, a greater functional specificity can beachieved by adding specific functional sequences to the bioelastomers ofthe present invention, as described above. This property is relevant tothe development of a surgical adhesion preventative biomaterial. Forexample, for cardiopulmonary by-pass procedures where there can besubstantially more bleeding than in the contaminated peritoneal cavitymodel, the bioelastomers can also provide a suitable non-adhesive matrixfor the testing of incorporated adhesion-promoting sequences in thepresence of serum without the background interference of serum proteinbinding resulting in cell adhesion. Another relevant application couldbe to form the intimal lining of a vascular prosthesis.

For example, by adding to the protein-based polymer a cell attachmentsequence such as Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) from fibronectin toresult in, for example, the elastic matrix X²⁰ -poly[40(GGAP),(GRGDSP)],a matrix which was refractory to cell adhesion now promotes celladhesion, cell spreading and growth to confluence. Importantly, thismeans that cells can migrate into and attach to the matrix and besubjected to, and also to sense the tensional forces to which the matrixis subjected in its functional role. It is now appreciated, for example,that the cyclic stretching to which a vascular wall is subjected inducesthe vascular wall cells to turn on the genes which result in elaborationof the macromolecules required to maintain and to rebuild the tissuerequired to sustain such tensional forces. This has been calledtensegrity, and it provides the basis for developing a temporaryfunctional scaffolding into which the natural cells can migrate, attach,and remodel into the natural required tissue.

The cell adhesion of X²⁰ -poly(GVGVP) in the presence of serum isprobably due to the association or absorption of adhesion proteins suchas fibronectin and vitronectin from the serum as occurs with otherbiomaterial. Equally, the lack of cell adhesion to the presentbioelastomers in the presence of serum would seem to be due to the lackof association or absorption of adhesion-promoting serum components.Although not wishing to be bound by a particular hypothesis, theinventor believes that the observed correlation of increased celladhesion with increased hydrophobicity of the polytetrapeptide-basedmatrices in the presence of serum suggests that the serum componentsresponsible may associate by hydrophobic interactions and that at acertain reduced level of matrix hydrophobicity, for example that of X²⁰-poly(GGAP), their association is insufficient to support cellattachment.

Another aspect of the invention is directed to a method forsubstantially preventing cellular adhesion by forming a protective layerbetween a first surface and a second surface using the presentbioelastomers. The surface referred to can be a tissue culture vesselmaterial, e.g. glass, plastic, or cells, tissue, dried body fluids atwound repair sites, or a mechanical implant such as a catheter, surgicaldrain, or shunt. The bioelastomer would generally form a layer of atleast 10 Å, preferably at least 10 Å, in thickness on the surface andcan optionally be linked to the surface, as described above.Alternatively one of the surfaces can be entirely formed from a materialof the invention.

Studies of the efficacy of various materials as a barrier in theprevention of adhesions employing a contaminated peritoneal modelindicates the utility of the polymers of the invention forcardio-pulmonary bypass products. With the propensity of suchbiopolymers to act innocuously in the body and to be refractory toformation of adhesions coating these polymers onto catheters, leads andtubings that would reside in the body for days, weeks and even monthswould result in a more ready removal and to do so with minimal damage totissues that would have been in contact with such temporary devices.

The invention now being generally described, the same will be betterunderstood by reference to the following examples, which are providedfor purposes of illustration only and are not to be considered limitingof the invention unless so specified.

EXAMPLES 1. Peptide Synthesis

The synthesis and cross-linking of poly(GVGVP) have been described inU.S. Pat. No. 4,783,523. The syntheses of poly(IPGG) has been describedin U.S. Pat. No. 5,250,516. The tetrapeptide monomer Boc-GGAP-OH wassynthesized by the stepwise solution phase method described below andthen formed into polymers.

In the Examples, the following abbreviations will be used: Boc,tertbutyloxycarbonyl; Bzl, benzyl; DMF, dimethylformamide; DMSO,dimethylsulfoxide; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;HOB, 1-hydroxybenzotriazole; IBCF, isobutyl-chloroformate;N-methylmorpholine; ONp, p-nitrophenylester; TFA, trifluoroacetic acid.All Boc amino acids and HOBt were purchased from Advanced ChemTech,(Louisville, Ky.). EDC was obtained from Bachere, Inc. (Torrance,Calif.).

Boc-Ala-Pro-OBzl (I)

Boc-Ala-OH (56.76 g, 0.3 mole) dissolved in acetonitrile (500 mL) andcooled to 0° C. before adding NMM (32.97 mL). The solution was cooled to-15°±1° C. and isobutyl chloroformate (41.01 mL) was added slowly understirring while maintaining the temperature at -15° C. After stirring thereaction mixture for 10 min at this temperature, HOBt (40.56 g, 0.3mole) was added. The reaction mixture was stirred for an additional 10min and a pre-cooled solution of HC; H-Pro-OBzL (72.5 g, 0.3 mole) andNMM (32.97 mL) in DMF (600 mL) were added slowly. After 20 min, the pHof the solution was adjusted to 8 by the addition of NMM and thereaction was continued overnight. The solvent was removed under reducedpressure and the residual DMF solution was poured into about 2000 mL ofice-cold 90% saturated KHCO₃ solution and stirred for 30 min. Since thepeptide did not precipitate out, it was extracted into CHCl₃, which waswashed with water, 20% citric acid, and water, and dried over Na₂ SO₄,and the solvent was washed with water, 20% citric acid, and water,before drying over Na₂ SO₄, and removing solvent under reduced pressure.The resulting oil was recrystallized from ether/petroleum ether. Thecrystals were filtered, washed with petroleum ether, and dried to obtain79.9 g (yield, 70.75 %) of I. R¹ _(f), 0.5; R² _(f) 0.61.

Boc-Gly-Ala-Pro-OBzl (II)

Boc-Ala-Pro-OBzl (75.3 g, 0.2 mole) was deprotected by stirring for 1.5h. in 4.2 N HCl in dioxane. Excess HCl and dioxane were removed underreduced pressure, triturated with ether, faltered, washed with ether,and dried (yield 100%). A solution of Boc-Gly-OH (35.04 g, 0.2 mole) andHOBt (27.02 g, 0.2 mole) in DMF was cooled to -15° C. with stirring andEDCI (38.34 g, 0.2 mole) was added. After 20 rain, a pre-cooled solutioncontaining the above hydrochloride salt and NMM (21.98 mL, 0.2 mole) wasadded and the reaction mixture was stirred overnight at roomtemperature. The mixture was evaporated to a thick oil which wasdissolved in CHCl₃. This solution was extracted with water, 10% citricacid, water, 5 % NaHCO₃, water, and dried over Na₂ SO₄. The solvent wasremoved under reduced pressure and the resulting oil was crystallizedfrom ether/petroleum ether. The solid was filtered, washed withpetroleum ether, and dried to obtain 76.2 g (yield 87.88%) of II. R¹_(f) 0.39; R² _(f), 0.44.

Boc-Gly-Gly-Ala-Pro-OBzl (III)

Compound II (65.03 g, 0.15 mole) was deblocked with HCl/dioxane andcoupled to Boc-Gly-OH (26.3 g, 0.15 mole) using EDCI with HOBt in thesame manner as that described for II to give 63.2 g (yield 85.9%) of IIIR¹ _(f), 0.3; R² _(f), 0.46.

Boc-Gly-Gly-Ala-Pro-ONp (IV)

Boc-Gly-Gly-Ala-Pro-OBzl (30.0 g, 0.061 mole) was dissolved in glacialacetic acid (300 mL) and 3.0 g of 10% Palladium on activated charcoalwas added. This mixture was hydrogenated at 40 psi for 6 h. The reactionmixture was filtered through celite and the solvent was removed invacuum. The resulting residue was triturated with ether, faltered,washed with ether, and dried to obtain Boc-Gly-Gly-Ala-Pro-OH.

This acid was dissolved in pyridine (200 mL) and reacted with Bis-PNPC(1.5 equiv.). When the reaction was complete, as monitored by TLC, thesolvent was removed under reduced pressure. The residue was taken intoCHCl₃ and washed with water, 10% citric acid, water, 5 % NaHCO₃, water,and dried over Na₂ SO₄. The solvent was removed under reduced pressure,triturated with ether, faltered, washed with ether, and dried to obtain18.3 g (yield 57.52%) of IV. R¹ _(f), 0.22; R² _(f), 0.38; R³ _(f),0.48.

Poly(Gly-Gly-Ala-Pro) (V)

Boc-Gly-Gly-Ala-Pro-ONp (18.0 g, 0.035 mole) was deblocked using TFA anda one-molar solution of the TFA salt in DMSO was polymerized for 14 daysusing 1.6 equiv. of NMM as base. The polymer was dissolved inpyrogen-free water, dialyzed using 3500 mol. wt. cut-off dialysis tubingand lyophilized. The product was base-treated with 1 N NaOH (2 equiv.per tetramer), dialyzed using 50 kD mol. wt. cut-off tubing for 8 daysand lyophilized to obtain 5.02 g (yield 51.5 %) of V.

To verify structure and purity, the intermediate and final products werechecked by TLC, carbon-13 NMR spectroscopy, and amino acid analysis. Thepresence of all requisite peaks and the absence of extraneous peaks arerequired to verify the synthesis. Thin layer chromatography (TLC) wasperformed on silica gel plates obtained from Whatman, Inc., with thefollowing solvent systems: R¹ _(f), CHCl₃ :CH₃ OH: CH₃ COOH; R² _(f),CHCl₃ :CH₃ COOH (90:10:3); R³ _(f), CHCl₃ :CH₃ OH: CH₃ COOH (85:15:3).The compounds on TLC plates were detected by UV light by spraying withninhydrin or chlorine/toluidine.

2. Preparation of the Cross-linked Matrices

Poly(GGVP) and poly(GGAP) were dissolved separately in pyrogen-freewater at a concentration of 600 mg/mL. The solutions were then placed ina mold and centrifuged with the temperature maintained at 25° C. for 1h. The temperature was then raised to 40° C. and centrifuged for 8 morehours. Poly(GGIP) was also dissolved in pyrogen-free water at aconcentration of 400 mg/mL. The solution was centrifuged with thetemperature maintained at 10° C. over a period of 5 h and 30° C. over aperiod of 15 h. The coacervate phase was then checked for uniformity. Ifthey did not have irregularities such as bubbles, they were thenγ-irradiated with a 20 Mrad dose of cobalt-60 radiation to form thecross-links which resulted in an insoluble matrix. The molds were thenopened in a laminar flow hood using sterile conditions and placed untiluse in PBS containing 100 u/mL penicillin, 100 μg/mL streptomycin, and2.5 μg/mL amphotericin-B at 4° C.

3. Cell Culture

Bovine ligamentran nuchae fibroblasts (LNFs) were cultured in Dulbecco'smodified Eagle medium containing 10% fetal bovine serum, 2 nML-glutamine, 0.1 mM nonessential amino acids, 100 u/mL penicillin, and100 μg/mL streptomycin as described by Long et al. in Biochem. Biophys,Acta. 968,300-311(1988).

Human umbilical vein endothelial cells (ItUVECs) at passage 1 werebought from Endotech Corporation, (Indianapolis, Ind.). Cells werecultured in Medium 199 containing 25 mM HEPES, 20% fetal bovine serum, 2nM L-glutamine, 13.2 μL/mL Endo-Ret HI-GF (Endotech) endothelial cellgrowth supplement, 100 u/mL penicillin, 100 μg/mL streptomycin, and 2.5μg/mL amphotericin-B. Tissue culture flasks for HUVECs were precoatedwith 2 % bovine gelatin (Sigma) at 7.5 μl/cm².

Both LNFs and HUVECs were passaged by trypsinization and were used forexperiment at passage 8 or earlier.

4. Adhesion Assay

The assay and apparatus has been described by Nicol et al., J. Biomed.Mater. Res., 26, 393-413 (1992). Cells were harvested using 0.05 %trypsin/0.53 mM EDTA (Gibco) and then treated with 0.2 mg/mL soybeantrypsin inhibitor. The cell density plated on the test matrices was 100cells/mm². Cell concentration was adjusted to 0.32×10⁵ /mL and 50 μLaliquots were plated into glass cylinders containing 50 μL ofappropriate medium over discs of the test matrices. Three replicateswere used for each experimental condition. The apparatus was incubatedfor 3 or 20 h to allow cell adhesion, after which the matrices weregently rinsed in PBS, pH 7.2, and then flexed in 3.5 9 paraformaldehydein PBS, pH 7.2. Phase contrast microscopy was then used to count andclassify the adherent cells. The area of one field of view counted was1.2 sq. mm. and five such fields were counted for each replicate. Thetotal area plated over was 15.9 sq. mm. for each replicate. Thus, 37 9of the total area plated was counted. The target plated cell density of100 cells/sq. mm. was chosen as suitable for individual cellrecognition, quantitation, and classification. To normalize variationsin the actual density of cells plated between experiments, the resultsare given as the percent of the appropriate positive control.

To examine the adhesion of LNFs and HUVECs to the cross-linkedelastomeric matrices alone, the cells were plated onto the test matricesin the absence of serum and in the presence of 0.1% BSA. Afterincubation for 3 h the matrices and control substrata were rinsed andthen fixed, as described above. It was found (FIG. 1) that neither celltype was able to adhere to any of the three polytetramer-based matricesand that cell adhesion to X²⁰ -poly(GVGVP) was extremely poor. The fewfibroblasts or endothelial cells which were found adherent to thematrices were rounded and had not spread.

In the presence of 10% fetal bovine serum, LNFs were found to adhere toX²⁰ -poly(GVGVP) at near control tissue culture substratum levels after3 h incubation (FIG. 2). Also, on these matrices the fibroblasts werelargely well spread. The fibroblasts failed to attach or spread on theX²⁰ -poly(GGAP) after 3 h in the presence of serum (FIG. 2). The resultsfor HUVECs plated in the presence of 20% fetal bovine serum (FIG. 2)were similar to those for LNFs except that the relative levels of cellattachment to X²⁰ -poly(GGVP) and X²⁰ -poly(GVGVP) were much less. Thelevel of attachment of HUVECs to X²⁰ -poly(GGIP) was only slightly lessthan that found for LNFs. No endothelial cell adhesion to X²⁰-poly(GGAP) was found (FIG. 2).

After 20 h of incubation in the presence of serum (FIG. 3), the resultsfound for both LNFs and HUVECs were essentially the same as for 3 hincubation except that the relative levels of cell attachment to thematrices were generally reduced. Again, for both LNFs and HUVECs, theX²⁰ -poly(GGAP) gave no support for cell adhesion.

The mean residue hydrophobicity of the polytetrapeptide matrices basedon the temperature of inverse temperature transition hydrophobicityscale are 37° C. for GGAP, 31.5° C. for GGVP, and 28° C. for GGIP. Thelower the temperature the greater the hydrophobicity. It can be seenfrom FIGS. 2 and 3 that, of the three polytetrapeptide-based matrices inthe presence of serum, the least hydrophobic (GGAP) showed the lowestlevels of cell adhesion and the most hydrophobic (GGIP) showed thehighest levels of cell adhesion. The correlation coefficients for theserelationships were -0.92, -0.98, -0.98, and -0.90 for 3 h and 20 h ofLNF and 3 h and 20 h of HUVEC attachment, respectively.

5. Biocompatibillty a. The Ames Mutagenicity Test

The Ames test uses five strains of Sahnonella typhimurium eachcontaining a specific mutation in the histidine operon. Without thepresence of histidine, these five genetically altered strains--TA98,TA100, TA1535, TA1537 and TA1538 will not grow. To study mutagenicity,these five strains are placed in a histidine-free medium where onlythose able to mutate back to the wild non-histidine dependent type willform colonies. The reversion rate is normally constant. Accordingly, ifthe introduction of a test article causes at least a two-fold increasein reversion rate, as compared to spontaneous reversion in the presenceof negative control (in this case saline), then the test article isconsidered mutagenic and therefore a possible carcinogen.

The test solution was a 4 mg/ml solution in 0.85% saline of poly(GGAP),equivalently poly(APGG). As seen in TABLE 1, the test article was notfound to be mutagenic in this study. In fact, the test article wasindistinguishable from the saline negative control. The positivecontrols were: Dexon for TA98, TA100 and TA1537; sodium azide forTA1535; 2-aminofluorene and 2-nitrofluorene for TA1538, and2-aminofluorene for TA1 00. Metabolic activation is necessary for2-aminofluorene to be mutagenic, therefore the 2-aminofluorene wastested on TA100 and TA1538 both with and without S-9 mix. The S-9 mix,which is a microsomal fraction from an appropriately induced rat liverhomogenate, was used to detect any mutagenic chemicals present that mayrequire metabolic biotransformation to become active mutagenic forms.The innocuous nature of poly(GGAP) becomes apparent when its reversionrates are compared to those of Dexon which is a sufficientlybiocompatible material for regular use as a biodegradable suturematerial.

b. Cytotoxicity--Agarose Overlay

Confluent monolayers of the L-929 mouse connective tissue cells weregrown in culture flasks. Minimum essential medium was prepared, thenplaced in the culture flasks and allowed to solidify over the cells toform the agarose overlay. A 0.1 ml sample of 40 mg/ml poly(GGAP) in0.90% saline was dosed neat on a 12.7 mm diameter filter disk which wasthen placed on the solidified overlay surface. A like filter disk with0.1 ml of 0.9 % sodium chloride USP solution was used as a USP negativecontrol. The positive control was placed in a 25 sq. cm flaskequidistant from the test article on the agarose surface. The flaskcontaining controls and test article was then sealed and incubated for24 hours at 37° C. After 24 hours the culture was examined. The testarticle showed no cell lysis or toxicity (a 0 mm zone of lysis) whilethe negative controls were non-toxic and the positive control was toxic(with a 9 mm zone of lysis). Thus poly(GGAP) was found to be non-toxicto L-929 mouse connective tissue cells.

c. Acute Systemic Toxicity

Five mice were weighed and then injected by an intravenous route with a40 mg/ml solution of poly(GGAP) in 0.85% saline at a dose of 50 ml/kg.Five other mice were similarly injected with control saline solution.The animals were then observed at 4, 24, 48 and 72 hours. Significantsigns of toxicity are mortality, body weight loss of more than 2 g inthree or more mice, convulsions or prostration. Slight signs of toxicitywould be lethargy or hyperactivity. The mice appeared normal; no signsof toxicity were observed for poly(GGAP).

d. Intracutaneous Toxicity

A 0.2 ml dose of 40 mg/ml solution of poly(GGAP) in 0.9% saline wasinjected by the intracutaneous route into five separate sites on theright side of the back of two rabbits, on the left side 0.9% sodiumchloride USP solution was injected as a control. At 24, 48 and 72 hoursthe injection sites of the animals were observed for signs of erythema(ER) or edema (ED). In the first study, slight irritation or toxicitywas observed. As even a slight irritation was unexpected., the study wasrerun at 5 mg/ml. As shown by the results in TABLE 2 neither irritationnor toxicity was observed. Neither erythema (ER) nor edema (ED) wasobserved.

e. Systemic Antigenicity Study

Solutions of poly(GGAP), equivalently poly(APGG), at a concentration of40 mg/ml volumes in a 0.9% NaCl test solution, were injectedintrapefitoneally at 10 ml/kg body weight three times a week, everyother day, until six induction injections were conducted on six guineapigs. Similarly, three additional guinea pigs were injectedintraperitoneally with 0.9% NaCl solution as the control condition. Tendays after the last intrapefitoneal injection, all of the guinea pigswere challenged by an intravenous injection of the poly(GGAP) solutionand then observed for any signs of antigenicity. Significant signs ofreaction would be face pawing, eye blinking, lethargy, convulsions andeven death. There were no significant reactions to the challengeinjection. Therefore, the test article solution would not be consideredantigenie in the guinea pig.

f. Dermal Sensitization Study (A Maximization Method)

Fifteen Hanley guinea pigs were used in this study. A 40 mg/ml solutionof poly(GGAP) in 0.9% NaCl was the test article. For Induction I, theten animals to be used were clipped, then received three rows ofintradermal injections, 2 per row, in a 2×4 cm area. The injections were0.1 ml of Freud's Complete Adjuvant (FCA), 0.1 ml of the test article,and 0.1 ml of a 1:1 suspension of the FCA and test article. The fiveanimals to be used as controls at the challenge phase were not induced.For Induction II, one week later the area was reclipped and a 10% sodiumlauryl sulfate suspension (SLS) in petrolatum was massaged into the skinto produce a mild acute inflammation. Any SLS suspension remaining after24 hours was removed. A 2×4 cm of a Whatman No. 3 MM filter paper,saturated with 0.3 ml of the test article, was topically applied andsecured with nonreactive tape and an elastic bandage wrapped around thetrunk of each animal. The patch was removed after 48 hours.

Next the follow-up challenge was performed at 12 days. The area wasagain clipped and a nonwoven cotton disk in a Hill Top Chamber@ wassaturated with 0.3 ml of the test article, topically applied and held inplace for a 24-hour period with semi-occlusive hypoallergenic adhesivetape and an elastic bandage wrapped around the trunk of the animal. Thearea was observed 24, 48, 72 and 96 hours after patch removal. The testarticle, poly(GGAP), did not cause delayed contact sensitization in theguinea pig (See TABLE 3). Note: "Background or artifactual reactions(0.5 score) were not counted as evidence of a sensitization response."{NAmSA®}. The 0.5 score occurred with the same frequency in the controlsas in the test animals.

g. Rabbit Pyrogen Study

A 0.9 gram portion of poly(GGAP) was reconstituted in 90 ml of sterilenonpyrogenic saline. A single dose of 10 ml/kg was intravenouslyinjected into the marginal ear vein of three rabbits. Rectaltemperatures were measured and recorded before injection and every hourfor 3 hours afterwards. A maximum rise of 0.1° C. was recorded with asum in the three animals of 0.2° C. whereas a summed rise of less than1.4° C. would still be considered non-pyrogenic. Accordingly, the testsolution was judged to be non-pyrogenic.

h. Lee-White Clotting Study

A 100 mg portion of poly(GGAP) was reconstituted in 10 ml of 0.9% NaClsolution to yield a 10 mg/ml test solution. 0.5 ml of the test solutionwas added to six siliconized tubes. These tubes were placed in a 37° C.heat block along with three siliconized tubes (without material) whichserved as the control. One ml of fresh canine blood was added to eachtube.

At timed intervals, the first tube in each set of three was tilted untilnearly horizontal. The tilting procedure was repeated at 30 secondintervals until the blood clotted. As seen in TABLE 4, the replicatemean is greater in each case for the test article, poly(GGAP), than forthe control. Clearly, poly(GGAP) does not shorten the clotting time.

i. Hemolysis Test In Vitro

A clot-free blood sample from a New Zealand White rabbit was collectedinto an EPTA vacuum tube on the day the test was performed. As usual,four tubes were used in this study: a negative control containing 10 mlof 0.9% sodium chloride USP solution (SC); a positive control containing10 ml of USP purified water (PW) and 2 tubes each containing 0.2 ml ofthe test article which is a solution of 40 mg/ml poly(GGAP) in 0.9 %sodium chloride and 10 ml SC containing 0.2 ml of the test article whichis again a solution of 4-0 mg/ml poly(GGAP) in 0.9% sodium chloride. Toeach robe 0.2 ml of rabbit blood was added. The robes were then coveredand gently inverted to mix, then placed in a 37° C. water bath for 1hour. After incubation, the tubes were again gently inverted and thesolutions were decanted into centrifuge robes to be centrifuged for 10minutes at 1000× g. From absorbance values taken at 545 nm the percenthemolysis was determined by the equation:

    Test article--SC negative control PW positive control X100-% Hemolysis(2)

The result in each case was 0% hemolysis; poly(GGAP) was found to benon-hemolytic. The results of the nine biocompatibility tests aresummarized in TABLE 5.

                  TABLE 1                                                         ______________________________________                                        Plate Incorporation Assay for the Ames Mutagenicity Test                      Test Article: X.sup.20 -poly(GGAP), Batch CG65PA                              Salmonella                                                                              TA98    TAIO0   TA1535 TA1537 TA1538                                typhimurium                                                                             Number of Revertant Colonies (Average of                            Tester Strains                                                                          Duplicate Plates)                                                   ______________________________________                                        Saline    82      180     14     8      6                                     (- control)                                                                   Saline test                                                                             57      172     9      6      6                                     article solution                                                              (undiluted)                                                                   Saline w/S-9                                                                            92      173     15     11     11                                    (- control)                                                                   Saline w/S-9                                                                            74      180     12     7      11                                    test article solu-                                                            tion (undiluted)                                                              Dexon 1 mg/ml                                                                           1048    1408    N/A    247    N/A                                   (+ control)                                                                   Dexon 1 mg/ml                                                                           1256    1048    N/A    1048   N/A                                   w/S-9                                                                         (+ control)                                                                   Sodium azide                                                                            N/A     N/A     2752   N/A    N/A                                   O.1 mg/ml                                                                     (+ control)                                                                   Sodium azide                                                                            N/A     N/A     3 176  N/A    N/A                                   O.1 mg/ml                                                                     w/S-9                                                                         (+ control)                                                                   2-nitrofluorene                                                                         N/A     N/A     N/A    N/A    2800                                  1 mg/ml                                                                       (+ control)                                                                   2-nitrofluorene                                                                         N/A     N/A     N/A    N/A    2240                                  w/S-9                                                                         (+ control)                                                                   2-aminofluorene                                                                         N/A     184     N/A    N/A    11                                    O.1 mg/ml                                                                     (+ control)                                                                   2-aminofluorene                                                                         N/A     1296 '  N/A    N/A    2816                                  w/S-9                                                                         (+ control)                                                                   ______________________________________                                          N/A = Not Applicable                                                         *Reports from North American Science Associates (NAmSA ®)                 In no case was there a twofold or greater increase in the reversion rate      of the tester strains in the presence of the test article solution.      

                  TABLE 2                                                         ______________________________________                                        USP Intracutaneous Toxicity Observations                                      Test Article: Poly(GGAP)                                                      ______________________________________                                        KEY                                                                           ER   =     ERYTHEMA      ED   =   EDEMA                                       0    =     None          0    =   None                                        1    =     Barely Perceptible                                                                          1    =   Barely Perceptible                          2    =     Well Defined  2    =   Well Defined                                3    =     Moderate      3    =   Raised 1 mm                                 4    =     Severe        4    =   Raised > 1 mm                               RESULTS:                                                                                  24 HOURS                                                                              48 HOURS  72 HOURS                                        Rabbit No.        ER     ED   ER   ED   ER   ED                               ______________________________________                                        69235   Test      0      0    0    0    0    0                                        Control   0      0    0    0    0    0                                69226   Test      0      0    0    0    0    0                                        Control   0      0    0    0    0    0                                ______________________________________                                        RATING (TEST - CONTROL)                                                       ______________________________________                                               0.0-0.5                                                                             Acceptable                                                              0.6-1.0                                                                             Slight                                                                  <1.0  Significant                                                      ______________________________________                                         *Reports from North American Science Associates (NAmSA ®)                 Date Prepared: 28-93                                                          Date Injected: 28-93                                                          Date Terminated: 2/11/93                                                      Comments: Not Applicable.                                                

                  TABLE 3                                                         ______________________________________                                        Guinea Pig Sensitization                                                      Dermal Reactions - Challenge                                                  Test Article: X.sup.20 -poly(GGAP), Batch CG65PA.                             Hours following patch removal                                                                                        96                                     Animal  24         48         72       Right                                  No./Group                                                                             Right Flank                                                                              Right Flank                                                                              Right Flank                                                                            Flank                                  ______________________________________                                        1 Test  0          0.5        0.5      0                                      2 Test  0          0.5        0.5      0                                      3 Test  0          0          0        0                                      4 Test  0          0          0        0                                      5 Test  0          0          0        0                                      6 Test  0.5        0.5        0.5      0                                      7 Test  0          0          0        0                                      8 Test  0          0          0        0                                      9 Test  0          0          0        0                                      10 Test 0          0          0        0                                      11 Test 0          0          0        0.5                                    12 Test 0          0          0        0                                      13 Test 0          0.5        0        0                                      14 Test 0          0          0        0                                      15 Test 0.5        0.5        0.5      0                                      ______________________________________                                         Right Flank = test article solution (as received)                        

                  TABLE 4                                                         ______________________________________                                        Lee-White Clotting Study                                                      Test Article: Poly(GGAP), Batch CG65PA                                        ______________________________________                                               Series                                                                 Tubes    A        B         Replicate Mean                                    ______________________________________                                        1        11.5     11.5      11.5                                              2        14.5     15.0      14.8                                              3        18.0     18.0       18.0*                                            ______________________________________                                        Control:                                                                      ______________________________________                                               Series                                                                 Tubes    A        B         Replicate Mean                                    ______________________________________                                        1         9.0      7.5       8.3                                              2        14.0     12.5      13.3                                              3        15.5     15.0       15.3*                                            ______________________________________                                         *Lee-White coagulation time                                                   NOTE: Time recorded in minutes.                                          

                  TABLE 5                                                         ______________________________________                                        Summary of Biocompatibility Test Results for Poly(GGAP):                      Preparation CG65PA*                                                           Test        Description                                                                              Test System                                                                              Results                                     ______________________________________                                        (1) Ames        Determine  Salmonella                                                                             non-                                          (Mutagenicity)                                                                            reversion rate                                                                           typhimurium                                                                            mutagenic                                     MG019       to wild type                                                                  of histidine-                                                                 dependent                                                                     mutants                                                       (2) Cytoxicity  Agarose    L-929 mouse                                                                            non-toxic                                     Agarose over-                                                                             overlay    fibroblast                                             lay MG030   determine                                                                     cell death                                                                    and zone of                                                                   lysis                                                         (3) Acute       Evaluate   Mice     non-toxic                                     Systemic    acute                                                             Toxicity    systemic                                                          TU012       toxicity from                                                                 an I.V. or                                                                    I.P. injection                                                (4) Intracutaneous                                                                            Evaluate   Rabbit   initial test:                                 Toxicity    local dermal        slight irrita-                                TU013       irritant or         tion                                                      toxic effects       retest - no                                               by injection        irritation                                (5) Systemic    Evaluate   Guinea Pigs                                                                            non-antigenic                                 Antigenicity                                                                              general                                                           TA085       toxicology                                                    (6) Sensitization                                                                             Dermal     Guinea Pigs                                                                            non-sensitiz-                                 (Maximization                                                                             sensitization       ing                                           Method)     potential                                                         TA006                                                                     (7) Pyrogenicity                                                                              Determine  Rabbit   non-                                          TU010       febrile             pyrogenic                                                 reaction                                                      (8) Clotting Study                                                                            Whole blood                                                                              Dog      Normal                                        TA038       clotting times      clotting time                             (9) Hemolysis   Level of   Rabbit blood                                                                           non-                                          CB037       hemolysis in        hemolytic                                                 the blood                                                     ______________________________________                                         *Reports from North American Science Associates (NAmSA ®) where the       detailed results are kept in their archives.                             

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A bioelastomer comprising at least 5 repeatingtetrapeptide or pentapeptide monomeric units of the formula:

    R.sub.1 PGR.sub.2 G.sub.n

wherein R₁ is a peptide-producing residue of alanine or glycine; P is apeptide-producing residue of proline; G is a peptide-producing residueof glycine; R₂ is a peptide-producing residue of glycine or alanine; andn is 0 or 1, wherein said bioelastomer is capable of undergoing aninverse temperature transition to form a regular β-turn-containingdynamic structure.
 2. The bioelastomer of claim 1, wherein saidbioelastomer is cross-linked.
 3. The bioelastomer of claim 1, whereinsaid bioelastomer is a homopolymer.
 4. The bioelastomer of claim 1,wherein said bioelastomer is a block or random copolymer comprising twoor more of said monomeric units.
 5. The bioelastomer of claim 1, whereinsaid bioelastomer further comprises up to 30% peptide-bond-formingmonomeric units other than said monomeric units.
 6. The bioelastomer ofclaim 1, wherein said R₁ is alanine.
 7. The bioelastomer of claim 1,wherein R₂ is glycine.
 8. The bioelastomer of claim 1, wherein aterminal end of said bioelastomer further comprises at least onepeptide-forming unit of lysine.
 9. The bioelastomer of claim 1, whereina terminal end of said bioelastomer contain at least one sulfhydrylgroup.
 10. The bioelastomer of claim 1, wherein said bioelastomer ispresent as a coating on a surface.
 11. The bioelastomer of claim 10,wherein said bioelastomer is covalently attached to said surface.